title: HPLC-ESI-MS/MS analysis of hemoglobin peptides in tryptic digests of dried-blood spot extracts detects HbS, HbC, HbD, HbE, HbO-Arab, and HbG-Philadelphia mutations.
abstract: BACKGROUND
  Hemoglobinopathies are mutations resulting in abnormal globin chain structure; some have clinically significant outcomes such as anemia or reduced lifespan. Five β-globin mutations are (c.20A>T, p.E6V), (c.19G>A, p. E6K), (c.79G>A, p.E26K), (c.364G>C, p.E121Q), and (c.364G>A, p.E121K), resulting in HbS (sickle-cell hemoglobin), HbC, HbE, HbD-Los Angeles, and HbO-Arab, respectively. One α-globin mutation is (c.[207C>G or 207C>A], p.N68K), resulting in HbG-Philadelphia.
  
  
  METHODS
  HPLC-ESI-MS/MS analysis of dried-blood spot (DBS) punches from newborns extracted with a trypsin-containing solution provides greater than 90% coverage of α-, β-, and γ-globin amino acid sequences. Because the (c.20A>T, p.E6V), (c.19G>A, p. E6K), (c.79G>A, p.E26K), (c.364G>C, p.E121Q), (c.364G>A, p.E121K), and (c.[207C>G or 207C>A], p.N68K) mutations generate globin peptides with novel amino acid sequences, detecting one of these peptides in DBS extracts is indicative of the presence of a hemoglobinopathy in the newborn.
  
  
  RESULTS
  The method described here can distinguish normal β-globin peptides from the mutant HbS, HbC, HbE, HbD-Los Angeles and HbO-Arab peptides, as well as normal α-globin peptide from the mutant HbG-Philadelphia peptide, allowing the identification of unaffected heterozygotes such as HbAS, and of compound heterozygotes such as HbASG-Philadelphia.
  
  
  CONCLUSIONS
  This HPLC-ESI-MS/MS analytical approach provides information that is not available from traditional hemoglobin analyses such as isoelectric focusing and HPLC-UV. It is also capable of determining the amino acid sequence of hemoglobin peptides, potentially allowing the detection of numerous hemoglobinopathies resulting from point mutations.
authors.affiliation: Biochemical Mass Spectrometry Laboratory, Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Mail Stop F-19, Atlanta, GA, 30341, United States. Electronic address: cph7@cdc.gov.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Haynes
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Guerra
authors.forename: Jessalyn C
authors.initials: JC
authors.lastname: Fontana
authors.forename: Víctor R
authors.initials: VR
authors.lastname: DeJesús
mesh_terms: D002851:Chromatography, High Pressure Liquid; D059788:Dried Blood Spot Testing; D006444:Hemoglobin C / Q000032:analysis / Q000235:genetics; D006446:Hemoglobin E / Q000032:analysis / Q000235:genetics; D006451:Hemoglobin, Sickle / Q000032:analysis* / Q000235:genetics; D006455:Hemoglobins, Abnormal / Q000032:analysis* / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D007231:Infant, Newborn; D009154:Mutation; D010455:Peptides / Q000032:analysis; D059748:Proteolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D006451:Hemoglobin, Sickle; D006455:Hemoglobins, Abnormal; D010455:Peptides; C032001:hemoglobin D; D006444:Hemoglobin C; D006446:Hemoglobin E; C031679:hemoglobin G Philadelphia; C032031:hemoglobin Arab; D014357:Trypsin
keywords: CID; DBS; Dried-blood spot; ESI; HPLC–ESI-MS/MS; Hb; Hemoglobinopathies; IEF; MALDI; SIM; collision-induced dissociation; dried-blood spot; electrospray ionization; hemoglobin; isoelectric focusing gel; matrix-assisted laser desorption ionization; single-ion monitoring